The present invention relates to methods and compositions for stimulating immune responses in mammals. More particularly, the invention relates to methods and compositions for stimulating mucosal immunity.
Many infectious pathogens, e.g., HIV-1, enter their mammalian hosts via a mucosal tissue prior to establishing a systemic infection. Veazey, et al., Science 280:427-431, 1998. Accordingly, vaccines capable of protecting against HIV should be capable of inducing long-term mucosal immune responses. A number of recent studies have shown that such immune responses require direct stimulation of mucosal tissues, and may be achieved with live attenuated virus, Cranage, et al., Virology 229:143-154, 1997, subunit SIV envelope Lehner, et al., Nature Medicine 2:767-775, 1996, HIV-recombinant viruses, including recombinant MVA 89.6 env (Belyakov et al., unpublished), or HIV peptide constructs Belyakov, et al., Proc. Nat. Acad. Sci. 95:1709-1714, 1998 (see also, Gallichan, et al., J. Exp. Med. 184:1879-1890, 1996; Cranage, et al., Virology 229:143-154, 1997; and Rosenthal, et al., Semin. Immunol. 9:303-314, 1997).
Numerous questions remain, however, concerning which vaccine candidates may afford the most effective protection against mucosal challenge with virus, and what mechanisms may be involved in mediating protective immunity. While a number of studies have shown a role for CTL in protection against infections such as influenza that have a mucosal component (Taylor and Askonas, Immunology 58:417-420, 1986; Epstein et al., J. Immunol. 160:322-327, 1998; Kulkarni et al., J. Virol. 69:1261-1264, 1995), these reports have not established whether the CTL need to be in a local mucosal site to protect. Conversely, while other studies have shown the induction of CTL in the mucosa, they have not established that these cells have a role in protection (Gallichan and Rosenthal, J. Exp. Med. 184:1879-1890, 1996; Bennink et al., Immunology 35:503-509, 1978; Lohman et al., J. Immunol. 155:5855-5860, 1995); and Klavinskis, et al., J. Immunol. 157:2521-2527, 1996 J. Immunol. 155:5855-5860, 1995. Yet other studies have shown the induction by vaccines of protective immunity in the mucosa, but in the face of multiple immune responses, have not been able to sort out which responses are involved in protection (Lehner et al., Nature Medicine 2:767-775, 1996; Putkonen et al., J. Virol. 71:4981-4984, 1997; Miller et al., J. Virol. 71:1911-1921, 1997; Quesada-Rolander et al., AIDS Res Hwn Retroviruses 12:993-999, 1996; Bender et al., J. Virol. 70:6418-6424, 1996; Wang et al., Vaccine 5:821-825, 1997).
Thus, although the role of CTL in protection against mucosal infections has been of interest for decades, especially in the case of influenza virus, prior investigations have failed to identify fundamental mechanisms linking immune responses to protection. In this regard, because mucosal infection by virus induces a local IgA response, it has been too readily assumed that this response, and not a concomitant CTL response, was responsible for protection against viral infection through the mucosal route. However, the role of secretary IgA in neutralizing and protecting against mucosal HIV challenge is also not clear.
CTL are crucial mediators of immunity to intracellular microorganisms such as viruses as well as certain bacteria and protozoan parasites. CTL specifically recognize xe2x80x9cnon-selfxe2x80x9d antigenic peptides bound to major histocompatibility complex (MHC) class I molecules on the surface of xe2x80x9ctarget cellsxe2x80x9d and then kill the target cells expressing the non-self antigenic peptides. Non-self polypeptides from which the non-self peptides are derived can be a) proteins encoded by intracellular microbes, b) host-encoded proteins whose expression is induced by a microbe, or c) mutant host encoded proteins expressed by, for example, tumor cells.
Thus, generation of CTL responses in the inductive and the effector mucosal immune system may be important to establishing effective protective immunity to intracellular microbial pathogens that establish infection via the mucosal barriers. In some cases, administration of antigens via parenteral routes (subcutaneous, intramuscular, intravenous or intraperitoneal, for example) either fails to induce mucosal immunity or does so extremely inefficiently.
As noted above, previous reports of mucosal immune responses elicited by mucosal challenge with viruses have disclosed that the latter induces antiviral antibody responses, and in some cases CTL responses, in the intraepithelial lymphoid populations. Chen et al., J. Virol. 71:3431-3436, 1997; Sydora, et al., Cell Inununol. 167:161-169, 1996. However, it is not clear if either of such responses is relevant to protection against viral infection in general, or HIV infection in particular. Additional studies have suggested a role for CTL in protection against infections that involve the mucosa, such as influenza or respiratory syncytial virus, Taylor et al., Immunology 58:417-420, 1986; Epstein et al., J. Immunol. 160:322-327, 1998; Kulkarni et al., J. Virol. 69:1261-1264, 1995. However, these studies have not addressed the question of whether CTL must be present at the mucosal site of infection, or if their principal activity occurs systemically.
Accordingly, a need exists in the art to better define the roles and mechanisms of CTL in mediating immunity and to develop new tools for mediating immune protection against HIV and other pathogens, particularly by conferring immune protection at mucosal sites where such pathogens initially proliferate.
The present invention is directed to methods and compositions for inducing a protective mucosal CTL response in a subject. The methods of the invention involve administering either a soluble antigen itself, or a polynucleotide encoding the soluble antigen, to a mucosal surface. The soluble antigens can be full length, naturally occurring polypeptides or fragments (i.e., peptides) derived from them. Peptides to be administered can be any length less than that of the naturally occurring polypeptide. They can be, for example, five to one hundred amino acid residues long, preferably twenty to seventy five amino acid residues long, more preferably twenty five to sixty amino acid residues long and most preferably thirty to fifty amino acid residues long.
The soluble antigen is administered with an adjuvant at the mucosal site or without an adjuvant. Adjuvants can be, for example, cholera toxin (CT), mutant CT (MCT), E. coli heat labile enterotoxin (LT) or mutant LT D1 (MLT). IL-12 and/or IFNxcex3 can be administered with the soluble antigen either in the presence or absence of an adjuvant. Alternatively, the two cytokines (IL-12 and/or IFNxcex3) can be administered systemically and separately from the soluble antigen which is administered mucosally, optionally with adjuvant. Mucosal routes of administration include IR, intranasal (IN), intragastric (IG), intravaginal (IVG) or intratracheal (IT).
Soluble antigens can be derived from pathogenic viruses (e.g., HIV-1, influenza virus or hepatitis A virus), bacteria (e.g, Listeria monocytogenes), protozoans (e.g., Giardia lamblia). Alternatively, the soluble antigen can be a tumor-associated antigen, e.g., prostate specific antigen produced by prostate tumor cells or tyrosinase produced by melanoma cells. Peptide antigens can be cluster peptide vaccine constructs (CL WvAC). For example, an HIV-1 CLUVAC can include one or more of the following sequences: EQMHEDIISLWDQSLKPCVKRIQRGPGRAFVTIGK (SEQ ID NO:1), KQIINMWQEVGKAMYAPPISGQIRRIQRGPGRAFVTIGK (SEQ ID NO:2), RDNWRSELYKYKVVKIEPLGVAPTRIQRGPGRAFVTIGK (SEQ ID NO:3), AVAEGTDRVIEVWQGAYRAIRHIPRRIRQGLERRIQRGPGRAFVTIGK (SEQ ID NO:4), DRVIEVVQGAYRAIRHIPRRIRQGLERRIQRGPGRAFVTIGK (SEQ ID NO:5), DRVIEVVQGAYRAIRRIQRGPGRAFVTIGK (SEQ ID NO:6), AQGAYRAIRHIPRRIRRIQRGPGPRAFVTIGK (SEQ ID NO:7), EQMHEDIISLWDQSLKPCVKRIHIGPGRAFYTTKN (SEQ ID NO:8), KQIINMWQEVGKAMYAPPISGQIRRIHIGPGRAFYTTKN (SEQ ID NO:9), RDNWRSELYKYKVVKIEPLGVAPTRIHIGPGRAFYTTKN (SEQ ID NO:10), AVAEGTDRVIEVVQGAYRAIRHIPRRIRQGLERRIHIGPGRAFYTTKN (SEQ ID NO:11), DRVIEVVQGAYRAIRHIPRRIRQGLERRIHIGPGRAFYTTKN (SEQ ID NO:12), DRVIEVVQGAYRAIRRIHIGPGRAFYTTKN (SEQ ID NO:13) or AQGAYRAIRHIPRRIRRIHIGPGRAFYTTKN (SEQ ID NO:14).
Preferably, the CLUVAC includes the amino acid sequence of SEQ ID NO:2, SEQ ID NO:9 or SEQ ID NO:12. Antigenic peptides can be longer than the length specified by the SEQ ID NOS.recited herein, i.e., the peptide can be extended by adding one or more (e.g., 5, 10, 15, 20) amino acids at the amino and/or carboxy termini of the peptide with any given SEQ ID NO.
The invention also encompasses methods for inducing a protective mucosal CTL response in a subject in which the soluble antigen is delivered IR. Preferably, the level of CTL activity induced by IR immunization is at least 10% greater than that induced by other routes of mucosal administration (e.g., IN). More preferably, mucosal CTL activity induced by IR immunization is at least 2-fold, more preferably, at least 5-fold, and most preferably, at least 10-fold greater than that induced by other routes of mucosal immunization (e.g., IN or IG).
Subjects to which the methods of the invention are applied are mammals, e.g., humans, non-human primates, cats or mice.
Also provided within the invention are immunogenic compositions for inducing a protective mucosal CTL response in a subject which are adapted for intrarectal administration. The compositions comprise a purified soluble antigen formulated for intrarectal delivery to the rectum, colon, sigmoid colon, or distal colon. They may be, formulated as a rectal enema, foam, suppository, or topical gel and generally comprise a base, carrier, or absorption-promoting agent adapted for intrarectal delivery.
In more detailed aspects, the immunogenic compositions of the invention may include a rectal emulsion or gel preparation, preferably wherein the soluble antigen is admixed with a homogenous emulsion or gel carrier, eg., a polyoxyethylene gel. Alternatively, the soluble antigen may be admixed with a rectally-compatible foam.
In other preferred aspects, the immunogenic compositions of the invention are formulated in a suppository. The suppository is comprised of a base or carrier specifically adapted for intrarectal delivery of the antigen. Preferred bases may be selected from a polyethyleneglycol, witepsol H15, witepsol W35, witepsol E85, propyleneglycol dicaprylate (Sefsol 228), Miglyol8lo, hydroxypropylcellulose-H (HPC), or carbopol-934P (CP). More preferably, the suppository comprises at least two base materials to optimize structural and delivery performance. In other aspects, the suppository includes a stabilizing agent to minimize intrarectal deradation of the soluble antigen.
To optimize intrarectal delivery, the immunogenic compositions of the invention also preferably include an absorption-promoting agent, for example a surfactant, mixed micelle, enamine, nitric oxide donor, sodium salicylate, glycerol ester of acetoacetic acid, cyclodextrin or beta-cyclodextrin derivative, or medium-chain fatty acid.
In yet additional aspects of the invention, immunogenic compositions are provided which include an adjuvant which enhances the CTL response. Suitable adjuvants are detoxified bacterial toxins, for example detoxified cholera toxin (CT), mutant cholera toxin (MCT), mutant- E. coli heat labile enterotoxin, and pertussis toxin. Preferably, the adjuvant is conjugated to a mucosal tissue or T cell binding agent, such as protein A, an antibody that binds a mucosal tissue- or T-cell-specific protein, or a ligand or peptide that binds a mucosal tissue- or T-cell-specific protein. In more preferred aspects, the adjuvant is a recombinant cholera toxin (CT) having a B chain of CT substituted by protein A conjugated to a CT A chain, which exhibits reduced toxicity and enhances mucosal tissue binding mediated by protein A. Alternatively the adjuvant may be conjugated to a protein or peptide that binds specifically to T cells, for example by binding CD4 or CD8 (eg., the HIV V3 loop or a T cell-binding peptide fragment of the HIV V3 loop).
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present application, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and not intended to be limiting other features and advantages of the invention, e.g., prevention of viral or other infectious diseases, will be apparent from the following detailed description, from the drawings and from the claims.